title: Structure and functional relevance of a transcription-regulating sequence involved in coronavirus discontinuous RNA synthesis.
abstract: Transmissible gastroenteritis coronavirus (TGEV) genomic RNA transcription generates 5'- and 3'-coterminal subgenomic mRNAs. This process involves a discontinuous step during the synthesis of minus-sense RNA that is modulated by transcription-regulating sequences located at the 3' end of the leader (TRS-L) and also preceding each viral gene (TRS-Bs). TRSs include a highly conserved core sequence (CS) (5'-CUAAAC-3') and variable flanking sequences. It has been previously proposed that TRS-Bs act as attenuation or stop signals during the synthesis of minus-sense RNAs. The nascent minus-stranded RNA would then be transferred by a template switch process to the TRS-L, which acts as the acceptor RNA. To study whether the TRS-L is structured and to determine whether this structure has a functional impact on genomic and subgenomic viral RNA synthesis, we have used a combination of nuclear magnetic resonance (NMR) spectroscopy and UV thermal denaturation approaches together with site-directed mutagenesis and in vivo transcriptional analyses. The results indicated that a 36-nucleotide oligomer encompassing the wild-type TRS-L forms a structured hairpin closed by an apical AACUAAA heptaloop. This loop contains most of the CS and is isolated from a nearby internal loop by a short Watson-Crick base-paired stem. TRS-L mutations altering the structure and the stability of the TRS-L hairpin affected replication and transcription, indicating the requirement of a functional RNA hairpin structure in these processes.
authors.affiliation: Centro de Investigación Príncipe Felipe, Avda. Autopista del Saler 16, 46012 Valencia, Spain.
authors.forename: David
authors.initials: D
authors.lastname: Dufour
authors.forename: Pedro A
authors.initials: PA
authors.lastname: Mateos-Gomez
authors.forename: Luis
authors.initials: L
authors.lastname: Enjuanes
authors.forename: José
authors.initials: J
authors.lastname: Gallego
authors.forename: Isabel
authors.initials: I
authors.lastname: Sola
mesh_terms: D020029:Base Pairing; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D012333:RNA, Messenger / Q000096:biosynthesis*; D012367:RNA, Viral / Q000096:biosynthesis*; D050436:Regulatory Elements, Transcriptional; D013056:Spectrophotometry, Ultraviolet; D014158:Transcription, Genetic; D005760:Transmissible gastroenteritis virus / Q000235:genetics* / Q000502:physiology
chemical_list: D012333:RNA, Messenger; D012367:RNA, Viral